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Loss of EP2 receptor subtype in colonic cells compromise epithelial barrier integrity by altering claudin-4.

Lejeune M, Moreau F, Chadee K - PLoS ONE (2014)

Bottom Line: Similarly, EP2 receptor antagonism/siRNA based silencing significantly decreased claudin-4 expression in EP2S cells.However, IFN-γ did not significantly decreased claudin-4 expression in Caco-2 cells indicating no role for IFN-γ in degrading claudin-4.We conclude that differential down-regulation of EP2 receptor play a major role in compromising colonic epithelial barrier function by selectively increasing proteosomal degradation of claudin-4.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
Prostaglandin E2 (PGE2) is a bioactive lipid mediator that exerts its biological function through interaction with four different subtypes of E-Prostanoid receptor namely EP1, EP2, EP3 and EP4. It has been known that EP2 receptor is differentially over-expressed in the epithelia of inflamed human colonic mucosa. However, the significance of the differential expression in altering epithelial barrier function is not known. In this study, we used Caco-2 cells expressing EP2 receptor, either high (EP2S) or low (EP2A), as a model epithelia and determined the barrier function of these cell monolayers by measuring the trans epithelial resistance (TER). Basal TER of EP2A (but not EP2S) monolayer was significantly lower suggesting a loss of colonic epithelial barrier integrity. In comparison, the TER of wild type Caco-2 was decreased in response to an EP2 receptor specific antagonist (AH-6809) indicating an important role for EP2 receptor in the maintenance of epithelial barrier function. The decrease TER in EP2A monolayer corresponded with a significant loss of the tight junction (TJ) protein claudin-4 without affecting other major TJ proteins. Similarly, EP2 receptor antagonism/siRNA based silencing significantly decreased claudin-4 expression in EP2S cells. Surprisingly, alteration in claudin-4 was not transcriptionally regulated in EP2A cells but rather undergoes increased proteosomal degradation. Moreover, among the TER compromising cytokines examined (IL-8, IL-1β, TNF-α, IFN-γ) only IFN-γ was significantly up regulated in EP2A cells. However, IFN-γ did not significantly decreased claudin-4 expression in Caco-2 cells indicating no role for IFN-γ in degrading claudin-4. We conclude that differential down-regulation of EP2 receptor play a major role in compromising colonic epithelial barrier function by selectively increasing proteosomal degradation of claudin-4.

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Pharmacological inhibition and/or siRNA silencing of EP2 receptor in EP2S cells decrease claudin-4 expression.(A) EP2S cells that reached confluence on regular culture plates were exposed with 50 µM of AH6809 (EP2 antagonist) for the time indicated and the cell lysate analyzed for claudin-4 expression by western blotting. Actin used as the internal control for the densitometry analysis. (B) EP2S cells seeded on culture plates that reached 60 percent confluence were transfected with either EP2 receptor or control siRNA. Cells collected after 48 h were checked for claudin-4 expression by western blotting and quantified by densitometry analysis for claudin-4 expression. ★ p<0.05; ★★ p<0.01 and ★★★ p<0.001.
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pone-0113270-g005: Pharmacological inhibition and/or siRNA silencing of EP2 receptor in EP2S cells decrease claudin-4 expression.(A) EP2S cells that reached confluence on regular culture plates were exposed with 50 µM of AH6809 (EP2 antagonist) for the time indicated and the cell lysate analyzed for claudin-4 expression by western blotting. Actin used as the internal control for the densitometry analysis. (B) EP2S cells seeded on culture plates that reached 60 percent confluence were transfected with either EP2 receptor or control siRNA. Cells collected after 48 h were checked for claudin-4 expression by western blotting and quantified by densitometry analysis for claudin-4 expression. ★ p<0.05; ★★ p<0.01 and ★★★ p<0.001.

Mentions: The results above suggest that cells lacking EP2 receptor expression was positively correlated with constitutive decrease in claudin-4 expression. To address specificity for this observation, we analyzed whether pharmacological inhibition or RNAi silencing of EP2 receptor could cause a similar decrease in claudin-4 expression. In EP2S cells, the EP2 receptor specific antagonist AH6809 (50 µM) decreased claudin-4 expression by 13 (p<0.05) and 32% (p<0.001) at 12 and 24 h, respectively (Fig. 5A). Similarly, in EP2S cells, siRNA silencing of EP2 receptor decreased claudin-4 expression by 24% within 48 h (p<0.001, Fig. 5B). These studies confirm that either inhibition of EP2 receptor activity or lack of EP2 receptor expression significantly decreased claudin-4.


Loss of EP2 receptor subtype in colonic cells compromise epithelial barrier integrity by altering claudin-4.

Lejeune M, Moreau F, Chadee K - PLoS ONE (2014)

Pharmacological inhibition and/or siRNA silencing of EP2 receptor in EP2S cells decrease claudin-4 expression.(A) EP2S cells that reached confluence on regular culture plates were exposed with 50 µM of AH6809 (EP2 antagonist) for the time indicated and the cell lysate analyzed for claudin-4 expression by western blotting. Actin used as the internal control for the densitometry analysis. (B) EP2S cells seeded on culture plates that reached 60 percent confluence were transfected with either EP2 receptor or control siRNA. Cells collected after 48 h were checked for claudin-4 expression by western blotting and quantified by densitometry analysis for claudin-4 expression. ★ p<0.05; ★★ p<0.01 and ★★★ p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4232557&req=5

pone-0113270-g005: Pharmacological inhibition and/or siRNA silencing of EP2 receptor in EP2S cells decrease claudin-4 expression.(A) EP2S cells that reached confluence on regular culture plates were exposed with 50 µM of AH6809 (EP2 antagonist) for the time indicated and the cell lysate analyzed for claudin-4 expression by western blotting. Actin used as the internal control for the densitometry analysis. (B) EP2S cells seeded on culture plates that reached 60 percent confluence were transfected with either EP2 receptor or control siRNA. Cells collected after 48 h were checked for claudin-4 expression by western blotting and quantified by densitometry analysis for claudin-4 expression. ★ p<0.05; ★★ p<0.01 and ★★★ p<0.001.
Mentions: The results above suggest that cells lacking EP2 receptor expression was positively correlated with constitutive decrease in claudin-4 expression. To address specificity for this observation, we analyzed whether pharmacological inhibition or RNAi silencing of EP2 receptor could cause a similar decrease in claudin-4 expression. In EP2S cells, the EP2 receptor specific antagonist AH6809 (50 µM) decreased claudin-4 expression by 13 (p<0.05) and 32% (p<0.001) at 12 and 24 h, respectively (Fig. 5A). Similarly, in EP2S cells, siRNA silencing of EP2 receptor decreased claudin-4 expression by 24% within 48 h (p<0.001, Fig. 5B). These studies confirm that either inhibition of EP2 receptor activity or lack of EP2 receptor expression significantly decreased claudin-4.

Bottom Line: Similarly, EP2 receptor antagonism/siRNA based silencing significantly decreased claudin-4 expression in EP2S cells.However, IFN-γ did not significantly decreased claudin-4 expression in Caco-2 cells indicating no role for IFN-γ in degrading claudin-4.We conclude that differential down-regulation of EP2 receptor play a major role in compromising colonic epithelial barrier function by selectively increasing proteosomal degradation of claudin-4.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
Prostaglandin E2 (PGE2) is a bioactive lipid mediator that exerts its biological function through interaction with four different subtypes of E-Prostanoid receptor namely EP1, EP2, EP3 and EP4. It has been known that EP2 receptor is differentially over-expressed in the epithelia of inflamed human colonic mucosa. However, the significance of the differential expression in altering epithelial barrier function is not known. In this study, we used Caco-2 cells expressing EP2 receptor, either high (EP2S) or low (EP2A), as a model epithelia and determined the barrier function of these cell monolayers by measuring the trans epithelial resistance (TER). Basal TER of EP2A (but not EP2S) monolayer was significantly lower suggesting a loss of colonic epithelial barrier integrity. In comparison, the TER of wild type Caco-2 was decreased in response to an EP2 receptor specific antagonist (AH-6809) indicating an important role for EP2 receptor in the maintenance of epithelial barrier function. The decrease TER in EP2A monolayer corresponded with a significant loss of the tight junction (TJ) protein claudin-4 without affecting other major TJ proteins. Similarly, EP2 receptor antagonism/siRNA based silencing significantly decreased claudin-4 expression in EP2S cells. Surprisingly, alteration in claudin-4 was not transcriptionally regulated in EP2A cells but rather undergoes increased proteosomal degradation. Moreover, among the TER compromising cytokines examined (IL-8, IL-1β, TNF-α, IFN-γ) only IFN-γ was significantly up regulated in EP2A cells. However, IFN-γ did not significantly decreased claudin-4 expression in Caco-2 cells indicating no role for IFN-γ in degrading claudin-4. We conclude that differential down-regulation of EP2 receptor play a major role in compromising colonic epithelial barrier function by selectively increasing proteosomal degradation of claudin-4.

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